Tracking mutant huntingtin aggregation kinetics in cells reveals three major populations that include an invariant oligomer pool

Abstract

Huntington disease is caused by expanded polyglutamine sequences in huntingtin, which procures its aggregation into intracellular inclusion bodies (IBs). Aggregate intermediates, such as soluble oligomers, are predicted to be toxic to cells, yet because of a lack of quantitative methods, the kinetics of aggregation in cells remains poorly understood. We used sedimentation velocity analysis to define and compare the heterogeneity and flux of purified huntingtin with huntingtin expressed in mammalian cells under non-denaturing conditions. Non-pathogenic huntingtin remained as hydrodynamically elongated monomers in vitro and in cells. Purified polyglutamine-expanded pathogenic huntingtin formed elongated monomers (2.4 S) that evolved into a heterogeneous aggregate population of increasing size over time (100-6,000 S). However, in cells, mutant huntingtin formed three major populations: monomers (2.3 S), oligomers (mode s(20,w) = 140 S) and IBs (mode s(20,w) = 320,000 S). Strikingly, the oligomers did not change in size heterogeneity or in their proportion of total huntingtin over 3 days despite continued monomer conversion to IBs, suggesting that oligomers are rate-limiting intermediates to IB formation. We also determined how a chaperone known to modulate huntingtin toxicity, Hsc70, influences in-cell huntingtin partitioning. Hsc70 decreased the pool of 140 S oligomers but increased the overall flux of monomers to IBs, suggesting that Hsc70 reduces toxicity by facilitating transfer of oligomers into IBs. Together, our data suggest that huntingtin aggregation is streamlined in cells and is consistent with the 140 S oligomers, which remain invariant over time, as a constant source of toxicity to cells irrespective of total load of insoluble aggregates.

FIGURE 1.

Hydrodynamic properties of recombinant Htt_ex1-Cerulean monomers. A, sedimentation velocity analysis of Htt_ex1^25Q-Cerulean and Htt_ex1^46Q-Cerulean at high speed (50,000 rpm). The initial data acquisition scans are shown in red and indicate all Htt_ex1-Cerulean to be in solution. The remaining scans at subsequent time points are shown in incrementing grayscale (note the scans may be overlapping). The data were fitted to a c(s) continuous size distribution, which best describes the behavior of small, diffusing particles (residuals to the c(s) fit are shown in the lower panels). The inset shows the sedimentation behavior of samples incubated for 72 h at 37 °C prior to data analysis. B, sedimentation coefficient distributions based on the fits in A for the c(s) analysis (top panel); note the overlapping data for the Htt_ex1^25Q-Cerulean samples. An alternate mode of analysis, the van Holde-Weischet analysis, which qualitatively describes the heterogeneity of sedimentation coefficients (lower panel) similarly indicates a population at 2–2.5 S.

FIGURE 2.

Kinetic assessment of aggregate size formed by recombinant Htt_ex1-Cerulean aggregates by low speed centrifugation (3,000 rpm). A, under these conditions, only large aggregates form sedimenting boundaries, whereas monomers do not sediment (resulting in a plateau). The first scan is shown in red, with subsequent scans in incrementing grayscale. The data for the 72-h incubated Htt_ex1^46Q-Cerulean were fitted to a ls-g*(s) continuous size distribution, which best describes the behavior of very large particles that have negligible diffusion (residuals to the fit shown in the inset). B, percentage of Htt_ex1^46Q-Cerulean partitioned in the sedimenting boundary after different incubation times, reflecting a transfer of monomers to aggregates over time. C, time-dependent changes in aggregate size heterogeneity and sedimentation coefficients of aggregated Htt_ex1^46Q-Cerulean were revealed by fits to ls-g*(s) size distributions at the different time points.

FIGURE 3.

Hydrodynamic assessment of Htt_ex1-Emerald monomers in Neuro2a lysates by sedimentation velocity analysis. A, Western blot shows the expression levels over 2 days. Only Htt_ex1^46Q-Emerald forms SDS-insoluble material over 2 days, indicative of aggregates. B, confocal microscopy shows Htt_ex1^25Q-Emerald to remain distributed evenly through the cytosol of Neuro2a cells after 2 days. In contrast, the Htt_ex1^46Q-Emerald forms punctate IBs in many cells. C, high speed sedimentation velocity analysis (50,000 rpm) of Htt_ex1-Emerald in the lysate (0.5 mg/ml total protein) after 2 days of expression. The first scan is shown in red, with subsequent scans in incrementing grayscale. The first scan shows a fast pile up of material at the bottom for Htt_ex1^46Q-Emerald for a fraction of the material. The lower panels show residuals to fits to a c(s) distribution. D, the sedimenting boundaries for Htt_ex1-Emerald dictate a highly monodisperse population (elongated monomers) when assessed by c(s) analysis (upper panel) or van Holde-Weischet analysis (lower panel). Zoomed in rescaling of the c(s) distributions shows a minor fraction of material to sediment at 3–8 S (inset of upper panel).

FIGURE 4.

Quantification of in-cell aggregate size heterogeneity with low speed and high viscosity (2 m sucrose) sedimentation velocity experiments (0.5 mg/ml total protein). A, sedimentation velocity analysis of the Htt_ex1^46Q-Emerald at low speed (3,000 rpm). The first scan is shown in red and in the absence of sucrose indicates a proportion of material that has already piled up on the bottom. The remaining sedimenting boundary (incrementing grayscale) was fitted to an ls-g*(s) size distribution (residuals to fit shown in the lower panels). Sedimentation was slowed by increasing the viscosity with 2 m sucrose. B, sedimentation velocity analysis of Htt_ex1^25Q-Emerald at low speed (3,000 rpm). Here, there is no sedimentation in the presence or absence of 2 m sucrose, indicative of a lack of aggregates. For comparison, lysates expressing β-galactosidase showed negligible signal intensity under the same photomultiplier voltage as Htt_ex1^25Q-Emerald (purple scans), indicating negligible nonspecific fluorescence contributions to the samples. C, ls-g*(s) size distributions detected at low speed (3,000 rpm) for the oligomer boundary detected in the absence of sucrose (left) and IBs from the boundary detected in 2 m sucrose (right). Data shown are the mean ± S.D. of fits to three independent preparations of lysates.

FIGURE 5.

The influence of Hsc70 on the evolution of three discrete species of Htt_ex1^46Q-Emerald in Neuro2a cells over 3 days of transient expression. A, the percentage of cells expressing Htt_ex1^46Q-Emerald containing IBs prior to lysis for assessment by SV analysis. Htt_ex1^46Q-Emerald was co-expressed with Hsc70-Emerald(Y66L) or β-galactosidase. Values are mean ± S.D. (error bars) (n = 3 separate transfections). B, the relative population of 2.3 S monomers, 140 S oligomers, and 320,000 S IBs as assessed by SV analysis (dashed line, Htt_ex1^46Q-Emerald and β-galactosidase; solid line, Htt_ex1^46Q-Emerald and Hsc70-Emerald(Y66L)). Values are mean ± S.D. (n = 3 separate transfections). C, the localization of Hsc70-Emerald and Htt_ex1-Cherry in Neuro2a cells after 3 days of transfection (imaged by confocal microscopy). Hsc70-Emerald is enriched at the periphery of the IBs in cells expressing Htt_ex1^46Q-Cherry and remains diffuse in cells expressing Htt_ex1^25Q-Cherry. Scale bar, 20 μm. D, Western blot probed with anti-GFP (top) and anti-α-tubulin (bottom) antibodies shows the expression levels and the presence of SDS-insoluble material over 3 days for the samples in A and B. Samples shown include Emerald and β-galactosidase (3-day expression; i), Hsc70-Emerald and β-galactosidase (3-day expression; ii), Htt_ex1^46Q-Emerald and β-galactosidase (iii), Htt_ex1^46Q-Emerald and Hsc70-Emerald(Y66L) (iv), and Htt_ex1^46Q-Emerald(Y66L) and Hsc70-Emerald (v). For all combinations, equivalent masses of DNA were used in the transfections. E, c(s) size distributions of high speed (50,000 rpm) SV analysis showed no detectable interactions between Hsc70-Emerald and Htt_ex1^46Q-Emerald(Y66L). F, ls-g*(s) size distributions of low speed (3,000 rpm) SV analysis showed no co-sedimentation of Hsc70-Emerald with Htt_ex1^46Q-Emerald(Y66L) 140 S oligomers or 320,000 S IBs. The first scan is shown in red, the last scan is shown in blue, and intermediate scans are shown in grayscale (note that many are overlapping due to lack of sedimentation).